Methods and kits for linking polymorphic sequences to expanded repeat mutations

Abstract

Methods and kits are provided for determining which single nucleotide polymorphism (“SNP”) variant of an allele of a heterozygous patient is on the same mRNA transcript as a disease-causing mutation that is at a remote region of the gene's mRNA comprising a) an allele-specific reverse transcription reaction using an allele-specific primer, and b) analysis of the resulting cDNA product from the reverse transcription reaction at the region of the mutation to determine the presence or absence of the mutation on this allele-specific cDNA product.

1 . A method for determining which single nucleotide polymorphism variant of an allele from a gene isolated from a heterozygous patient is on the same mRNA transcript as a disease-causing mutation at a remote region of the gene's mRNA comprising:
 a) an allele-specific reverse transcription reaction using an allele-specific primer which recognizes one single nucleotide polymorphism variant, and   b) analysis of an allele-specific cDNA product from the allele-specific reverse transcription reaction at the remote region of the gene to determine the presence or absence of the mutation on the allele-specific cDNA product.   
     
     
         2 . The method of  claim 1 , wherein the 3′ end of the allele-specific primer is positioned at the single nucleotide polymorphism nucleotide position. 
     
     
         3 . A method of  claim 1  further comprising:
 c) at least a second allele-specific reverse transcription reaction using at least a second allele-specific primer which recognizes at least a second single nucleotide polymorphism variant, and   d) analysis of at least a second allele-specific cDNA product from at least the second allele-specific reverse transcription reaction at the remote region of the gene to determine the presence or absence of the mutation on at least the second allele-specific cDNA product.   
     
     
         4 . A method of  claim 2  further comprising:
 c) at least a second allele-specific reverse transcription reaction using at least a second allele-specific primer which recognizes at least a second single nucleotide polymorphism variant, and   d) analysis of at least a second allele-specific cDNA product from at least the second allele-specific reverse transcription reaction at the remote region of the gene to determine the presence or absence of the mutation on at least the second allele-specific cDNA product.   
     
     
         5 . The method of  claim 3  wherein the 3′ end of at least the second allele-specific primer is positioned at the single nucleotide polymorphism nucleotide position. 
     
     
         6 . The method of  claim 4  wherein the 3′ end of at least the second allele-specific primer is positioned at the single nucleotide polymorphism nucleotide position. 
     
     
         7 . A method of treating a patient comprising:
 a) determining which single nucleotide polymorphism variant is on the same mRNA transcript as a disease causing mutation according to the method of  claim 1 , and   b) applying an allele-specific therapy to the single nucleotide polymorphism variant.   
     
     
         8 . The method of  claim 7 , wherein the allele-specific therapy comprises allele-specific RNA interference using siRNA or shRNA. 
     
     
         9 . The method of  claim 1 , wherein the patient is at risk of developing a neurodegenerative disorder selected from the group consisting of Dystophia myotonica, Spinocerebellar ataxia type 1, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3, Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, Spinocerebellar ataxia type 8, Spinocerebellar ataxia type 17, Huntington disease-like 2, Spinal and bulbar muscular atrophy, Huntington's disease, Dentatorubral-pallidoluysian atrophy, Oculopharyngeal dystrophy, Congenital central hypoventilation syndrome, Infantile spasms, Synpolydactyl), Cleidocranial dysplasia, Holoprosencephaly, Hand-foot-genital syndrome, Type II blephorophimosis, ptosis, and epicanthus inversus syndrome. 
     
     
         10 . The method of  claim 7 , wherein the allele-specific therapy operates at the same single nucleotide polymorphism site used to determine which mRNA transcript contains the disease-causing allele in said patient. 
     
     
         11 . The method of  claim 7 , wherein the allele-specific therapy operates at a different single nucleotide polymorphism site than the site used to determine which mRNA transcript contains the disease-causing allele in said patient. 
     
     
         12 . A kit for determining which single nucleotide polymorphism variant of an allele of a heterozygous patient is on the same mRNA transcript as a disease-causing mutation located at a remote region of the gene's mRNA comprising
 a) an allele-specific primer which recognizes one single nucleotide polymorphism variant, and   b) a set of instructions.   
     
     
         13 . The kit of  claim 12 , further comprising: at least a second allele-specific primer which recognizes at least a second single nucleotide polymorphism variant. 
     
     
         14 . The kit of  claim 12 , wherein the 3′ end of the allele-specific primer is positioned at the single nucleotide polymorphism nucleotide position. 
     
     
         15 . The kit of  claim 12 , wherein the 3′ end of at least the second allele-specific primer is positioned at the single nucleotide polymorphism nucleotide position. 
     
     
         16 . The kit of  claim 13 , wherein the 3′ end of at least the second allele-specific primer is positioned at the single nucleotide polymorphism nucleotide position. 
     
     
         17 . The kit of  claim 14 , wherein the 3′ end of at least the second allele-specific primer is positioned at the single nucleotide polymorphism nucleotide position. 
     
     
         18 . The kit of  claim 12 , wherein the allele-specific primer is selected from the group consisting of SEQ. ID. 1-SEQ. ID. 84. 
     
     
         19 . The kit of  claim 13 , wherein
 at least the second allele-specific primer is selected from the group consisting of SEQ. ID. 11-SEQ. ID. 20 if the allele-specific primer is selected from the group consisting of SEQ. ID. 1-SEQ. ID. 10; or   at least the second allele-specific primer is selected from the group consisting of SEQ. ID. 31-SEQ. ID. 40 if the allele-specific primer is selected from the group consisting of SEQ. ID. 21-SEQ. ID. 30; or   at least the second allele-specific primer is selected from the group consisting of SEQ. ID. 53-SEQ. ID. 64 if the allele-specific primer is selected from the group consisting of SEQ. ID. 41-SEQ. ID. 52; or   at least the second allele-specific primer is selected from the group consisting of SEQ. ID. 75-SEQ. ID. 84 if the allele-specific primer is selected from the group consisting of SEQ. ID. 65-SEQ. ID. 74.   
     
     
         20 . The kit of  claim 18 , wherein the allele-specific primer is selected from the group consisting of SEQ. ID. 42, SEQ. ID. 47, SEQ. ID. 52, SEQ. ID. 54, SEQ. ID. 59, and SEQ. ID. 64.